The present invention concerns methods and compounds for changing (modulating) tissue-remodeling processes.
The eukaryotic protein kinase superfamily is composed of enzymes which specifically phosphorylate serine, threonine or tyrosine residues of intracellular proteins. These enzymes are important in mediating signal transduction in multicellular organisms and are involved in a wide variety of cellular events. A few examples include: cellular proliferation, cellular differentiation, oncogenesis, immune responses, and inflammatory responses.
Enhanced protein kinase activity can lead to persistent stimulation by secreted growth factors and other growth inducing factors which, in turn, can lead to proliferative diseases such as cancer, to nonmalignant proliferative diseases such as arteriosclerosis, psoriasis and to inflammatory responses such as septic shock. Decreased function can also lead to disease. For example, a decrease in the activity of insulin receptor kinase is a cause of various types of diabetes.
Thus, agents which can modulate (increase or decrease) the activity of protein kinases have great potential for the treatment of a wide variety of diseases and conditions such as cancer, autoimmune disorders, and inflammation.
PKs are known to have homologous xe2x80x9ccatalytic domainsxe2x80x9d which are responsible to the phosphorylation activity. Based on a comparison of a large number of protein kinases, it is now known that the kinase domain of protein kinases can be divided into twelve subdomains. These are regions that are generally uninterrupted by large amino acid insertions and contain characteristic patterns of conserved residues (Hanks and Hunter, xe2x80x9cThe Eukaryotic Protein Kinase Superfamilyxe2x80x9d, in Hardie and Hanks ed., The Protein Kinase Facts Book, Volume I, Academic Press, Chapter 2, 1995). These subdomains are referred to as Subdomain I through Subdomain XII.
Due to the high degree of homology found in the subdomains of different protein kinases, the amino acid sequences of the domains of different PKs can be aligned. Frequently, the alignment is carried out with reference to the prototypical protein kinase PKA-Cxcex1, as known in the art. Currently, the catalytic domains of a large number of protein kinases have been aligned and tables showing these alignments are available from various published sources, such as, for example, in the article by Hanks and Quinn in Methods of Enzymology 200: 38-62 (1991) or in the PKR Web Site: www.sdsc.edu/kinases.
U.S. Pat. No. 6,174,993, International Application WO 98/53051 (corresponding to pending U.S. Application 08/861,153), International Application WO 00/118895 (corresponding to U.S. Ser. No. 09/161,094, now abandoned), U.S. applications Ser. No. 09/458,491, now abandoned (corresponding to WO 01/42280), and U.S. Ser. No. 09/734,520, now abandoned (corresponding to WO 02/48336) (all incorporated herein by reference) concern small, previously undisclosed, regions of various protein kinases with high substrate specificity. Short peptides derived from these regions were found to modulate kinase activities, as determined by the modulation of cellular activity in various in vivo and in vitro models. Without wishing to be bound by theory it is assumed that the short peptides disclosed in these applications, mimic some region of the catalytic domain of the kinase, bind to other cellular components with which the kinase interacts (such as the substrates of the kinase, other kinases, other phosphatases) and thus modulate kinase activity either by mimicking the kinase activity, or alternatively by inhibiting the interaction of the kinase and the cellular components thus inhibiting kinase-mediated signaling.
U.S. Pat. No. 6,174,993 and WO 98/53051 disclose a domain termed the HJ-loop. The xe2x80x9cHJ-loopxe2x80x9d referred to therein is found within the kinase domain of protein kinases between the middle of Subdomain IX and the middle of Subdomain X. Because of the high degree of homology found in the subdomains of different protein kinases, the amino acid sequences of the domains of different ser/thr protein kinases can be aligned. Thus, the HJ-loop of protein kinases can be defined by reference to the amino acid sequence of a prototypical protein kinase, for example PKA-Cxcex1, and can be said to correspond to a contiguous sequence of about twenty amino acid residues found between about amino acid 229 and 248 of PKA-Cxcex1.
A second definition of the HJ loop of protein kinases, which is complementary to the definition provided in the proceeding paragraph, can be made by reference to the secondary structure of the kinase domain of protein kinases. The kinase domain of protein kinases has been found to contain at least nine alpha helices, referred to as helix A through helix I, nine beta sheets, referred to as b1 through b9 (Tabor et al., Phil. Trans. R. Soc. Lond. B340: 315 (1993), Mohammadi et al., Cell 86:577 (1996) and Hubbard et al., Nature 372:746 (1994)). The HJ loop is a contiguous sequence of about twenty amino acids beginning within the F helix about five amino acids residues from the N-terminus of the F helix and extending about five amino acid residues into the G helix. It is noteworthy that the HJ-loop of the TGF-xcex2/ILK family of protein kinases contains an insertion of about 12 to 15 extra amino acids as compared to other ser/thr or tyrosine (tyr) protein kinases.
WO 02/48336 discloses a region termed the xe2x80x9cA-regionxe2x80x9d. The xe2x80x9cA-regionxe2x80x9d referred to herein is found within the kinase domain of PKs in Subdomain III and Subdomain IV. With respect to the amino acid sequence of the prototypical protein kinase PKA-Cxcex1 the A region can be said to correspond to a contiguous sequence of about eighteen amino acid residues found between about amino acids 92 and 109 of PKA-Cxcex1. In some PKs, extra amino acids can be present in this region and the size of the A-region can, therefore, include more than 18 amino acids in length.
With respect to the secondary structure of protein kinases, the A region is a contiguous sequence of about five to twenty amino acids beginning at the middle of the xcex1C helix (hereby xcex1C) and ending at the beginning of the b4 beta sheet.
WO 01/42280 discloses a region termed B4-B5 region. The xe2x80x9cB4-5 regionxe2x80x9d referred to herein is found within the kinase domain of PKs in Subdomain IV and the beginning of Subdomain V. With respect to the amino acid sequence of the prototypical protein kinase PKA-Cxcex1, the B4-5 region can be said to correspond to a contiguous sequence representing the amino acid residues found between about amino acids 106 and 114 of PKA-Cxcex1.
In some PKs, extra amino acids might be inserted in this region and the size of the B4-5 region can, therefore, include more than 9 amino acids in length.
A second definition of the B4-5 region of a PK, which is complementary to the definition provided in the preceding paragraph, can be made by reference to the three dimensional structure of the kinase domain of PKs. The kinase domain of PKs has been found to contain at least nine alpha helices, referred to as helix A through helix I and nine beta sheets, referred to as b1 through b9 (Tabor et al., Phil. Trans. R. Soc. Lond., B340: 315 (1993), Mohammadi et al., Cell, 86:577 (1996) and Hubbard et al., Nature 372:746 (1994). The B4-5 region is a contiguous sequence of about five to twenty five amino acids beginning at the end of the b4 beta sheet and into the b5 beta sheet.
WO 00/18895 discloses a region termed the xe2x80x9cxcex1D regionxe2x80x9d. The xe2x80x9cxcex1D regionxe2x80x9d referred to herein is found within the kinase domain of PKs in Subdomain V and the beginning of Subdomain VI. The xe2x80x9cxcex1D regionxe2x80x9d of a PK can be defined by reference to the amino acid sequence of a prototypical protein kinase, for example PKA-Cxcex1 and can be said to correspond to a contiguous sequence of about twenty amino acid residues found between about amino acid 120 and 139 of PKA-Cxcex1.
In relation to the secondary structure of the kinase domain of PKs, the xcex1D region is a contiguous sequence of about fifteen to forty amino acids beginning at the end of the b5 beta sheet and extending through the D helix and the following loop to the beginning of helix E.
The three dimensional structure of a number of kinases has been determined. A classical view of this structure is given in Knighton et al., Science 253, 407-414 (1991).
The superfamily of transforming growth factor beta (TGF-xcex2), which include TGF-xcex2, activins, and bone morphogenetic proteins (BMPs), are secreted agents that regulate a plurality of cellular responses such as proliferation, differentiation, migration and apoptosis.
TGF-xcex2 superfamily signaling has been implicated in a multitude of disorders and in various human diseases, including: cancer, fibrosis and autoimmune diseases. TGF-xcex2 superfamily members transduce their signals across the plasma membranes by inducing the formation of heteromeric complexes of specific type I and type II serine/threonine kinase receptors, which in turn activate a particular subset of SMAD proteins (some of them being inhibitory and some being excitatory). The SMAD molecule compounds relay the signals into the nucleus where they direct transcriptional responses in concert with other proteins.
TGF-xcex2 superfamily was found to be involved in embryogenesis-related physiological functions such as: regulation of growth, cell-faith specification, differentiation and apoptosis; in postnatal mechanisms including inhibition of cell growth (epithelial, vascular endothelial, hematopoietic cells and lymphocytes), induction of synthesis of IgA in B-lymphocytes, in control of wound healing processes and remodeling; in BMP-dependent responses including induction: of fibroblasts, osteoblasts in the process of cartilage and bone formation, respectively, involvement in bone genesis and spermatogenesis.
Malfunction connected with TGF-xcex2 has been linked to several clinical disorders including cancer, fibrosis, bone diseases, diabetic nephropathy, primary pulmonary hypertension, as well as chronic vascular diseases such as artheriosclerosis and hypertension.
There have been attempts to try and modify the process of bone healing by changing the concentrations of the TGF-xcex2 ligands. For example, various attempts have been made to increase the rate of bone healing by locally administrating to the fracture and of various TGF-xcex2 and BMP ligands, as well as by application of other growth factors. However, to date the results of these attempts have not been successful.
The present invention is based on the discovery that administration to the tissue of various compounds, which comprise peptides, having either a sequence appearing in one of four specific regions of TGF-xcex2 superfamily serine/threonine kinase receptor, or a variant of said sequence, resulted in a marked alteration of tissue remodeling processes in the tissue to which the compound was administered.
Without wishing to be bound by theory, it is assumed that the peptide part of the compound, mimics a region in the kinase that interacts with other cellular components such as the substrates of the kinase; phosphatases or other kinases which regulate the level of phosphorylation of the TGF-xcex2 kinase. The peptide part of the compound thus interrupts the interaction of the native kinase with the other cellular components (for example the substrates). Where the interaction between the kinase and the cellular component is an xe2x80x9conxe2x80x9d reaction, i.e., having a net result of the increase of physiological property such as increased transcription (for example interaction of the TGF-xcex2 kinase with an excitatory SMAD), the effect of the interruption of the xe2x80x9conxe2x80x9d reaction is inhibition of the property, for example inhibition of collagen synthesis. Where the interaction between the kinase and the cellular component is an xe2x80x9coffxe2x80x9d reaction, i.e., has a net result of decrease of physiological property, for example, decrease of transcription (such as interaction of TGFxcex2 with inhibitory SMAD), the interruption of the xe2x80x9coffxe2x80x9d reaction results in increased physiological property such as increased transcription leading to increased bone healing.
Thus, the present invention concerns a method for the modulation of tissue-remodeling comprising: contacting the tissue to be remodeled with an effective amount of a compound comprising a sequence selected from:
(a) a sequence which is a continuous stretch of at least five amino acids present in a native TGF-xcex2 super family Ser/Thr kinase receptor, in positions of the receptor corresponding to positions 249 to 279 of TGF-xcex2I receptor (HJ loop);
(b) a sequence which is a continuous stretch of at least five amino acids present in a native TGF-xcex2 super family Ser/Thr kinase receptor, in positions of the receptor corresponding to positions 119 to 139 of TGF-xcex2I receptor (xcex1D region);
(c) a sequence which is a continuous stretch of at least five amino acids present in a native TGF-xcex2 super family Ser/Thr kinase receptor, in positions of the receptor corresponding to positions 104 to 115 of TGF-xcex2I receptor (B4-B5 region);
(d) a sequence which is a continuous stretch of at least five amino acids present in a native TGF-xcex2 super family Ser/Thr kinase receptor, in positions of the receptor corresponding to positions 89 to 103 of TGF-xcex2I receptor (A-region);
(e) a variant of a sequence according to any one of (a) to (d) wherein up to 40% of the amino acid of the native sequence have been replaced with a naturally or non-naturally occurring amino acid or with a peptidomimetic organic moiety; and/or up to 40% of the amino acids have their side chains chemically modified; and/or up to 20% of the amino acids have been deleted; provided that at least 50% of the amino acids in the parent sequence of (a) to (d) are maintained unaltered in the variant, and provided that the variant maintains the biological activity of the parent sequences of (a) to (d);
(f) a sequence of any one of (a) to (e) wherein at least one of the amino acids is replaced by the corresponding D-amino acid;
(g) a sequence of any one of (a) to (f) wherein at least one of the peptidic backbones has been altered to a non-naturally occurring peptidic backbone;
(h) a sequence being the sequence of any one of (a) to (g) in reverse order; and
(i) a combination of two o r more of the sequences of (a) to (h)
The term xe2x80x9ctissue-remodelingxe2x80x9d, in the context of the present invention, refers to a wide variety of cellular and tissue-related processes which are characterized by a generation of new cells or tissues and/or degeneration of existing cells or tissues, or characterized by a change in the ratio between generation and degeneration of cells or extracellular matrix, which leads to a change in the entire tissue manifested by: increased tissue growth, decreased tissue growth, prevention of tissue formation, enhanced tissue healing, change in the proportion of various cell types in the tissue, change in the level of extracellular matrix in the tissue, etc. In particular, this term refers to, but is not limited to, bone remodeling (and in particular bone healing); skin-remodeling including in particular skin healing and prevention of scar formation in the skin; change of amount collagen deposition and in particular decrease of fibrosis formation, resulting in decreased scarring, and improved wound healing after surgery or injury, as well as decrease of post-surgery adhesions; change of cell proliferation and myogenic differentiation. This term includes, but is not limited to, skin remodeling, skin healing, scar formation, prevention of alopecia, improved hair growth, inhibition of fibrosis, modulation cell migration (including neuronal crest cell migration), modulating cell proliferation, modulating adipose tissue proliferation, and modulating myogenic differentiation.
The term xe2x80x9cmodulationxe2x80x9d refers to a change (increase or decrease) in the level of tissue-remodeling, as compared to the level of tissue-remodeling in the absence of the compound of the invention or in the presence of a control compound. The change should be statistically significant. The change of level can be determined in accordance with each tissue remodeling phenomena, for example, modulation of bone healing (which is a tissue remodeling process) can be determined by decrease in the time required until complete bone healing, by measurement of the size of callous formation, by determination of the amount of the normal bone produced in a set period of time, etc. Modulation in connection with fibrosis may be carried out by comparing the size of fibrous tissue in a test as compared to control, by comparing the amount of collagen deposition in test vs. control, etc.
Modulation of an individual""s tissue-remodeling refers to, for example, an inhibition of alopecia, enhancement of bone growth, prevention of fibrosis and scar formation, prevention of adipose cell proliferation, prevention of excess fibrosis formation in a plurality of diseases such as pulmonary fibrosis, chronic renal disease, scleroderma, liver cirrhosis, keloid formation, post-surgical adhesions and the like.
As will be explained later in detail, while direct assessment of tissue remodeling has to be carried out in tissue or in vivo, a good indication of tissue remodeling processes and modulation of said processes can be achieved by assessing the level of TGF-xcex2-associated signal transduction in cells (for example by determination of TGF-xcex2-dependent proliferation of fibroblasts) or in cell-free assays (such as by determination of the level of phosphorylation of the TGF-xcex2-kinase substrates).
The term xe2x80x9ccompound (comprising sequence)xe2x80x9d refers to a compound that includes therewithin any of the sequences of (a) to (i) as defined above. The compound may be composed mainly from amino acid residues, and in that case the amino acid component of the compound should comprise no more than a total of about 35 amino acids. Where the compound is mainly an amino acid compound, it may consist of any one of the amino acid sequences of (a) to (h), a combination of two or more, preferably of three most preferably of two, of the sequences of (a) to (h) linked to each other (either directly or via a spacer moiety) to give the combination of (i). The compound may further comprise any one of the amino acid sequences, or combinations as described above (in (a) to (i) above), together with additional amino acids or amino acid sequences. The additional amino acids may be sequences from other regions of the TGF-xcex2-kinase, sequences that are present in the kinase vicinity of the regions, N-terminal or C-terminal to the sequences of (a) to (d), or sequences which are not present in the TGF-xcex2-kinase but were included in the compound in order to improve various physiological properties, such as penetration into cells (sequences which enhance penetration through membranes or barriers); decrease degradation or clearance; decrease repulsion by various cellular pumps; improve immunogenic activities; improve various modes of administration (such as attachment of various sequences which allow penetration through various barriers, blood brain barrier, through the gut, etc.); increase specificity; increase affinity; decrease toxicity; and the like. A specific example is the addition of the amino acid Gly to the N-terminal of the sequence.
The compound may also comprise non-amino acid moieties, such as, for example, hydrophobic moieties (various linear, branched, cyclic, polycyclic or heterocyclic hydrocarbons and hydrocarbon derivatives) attached to the peptides of (a) to (i) to improve penetration, various protecting groups, especially where the compound is linear, attached to the compound""s terminals to decrease degradation; chemical groups present in the compound to improve penetration or decrease toxic side effects; or various spacers, placed, for example, between one or more of the above amino acid sequences, so as to spatially position them in a suitable orientation in respect of each other. The compound of the invention may be linear or cyclic, and cyclization may take place by any means known in the art. Where the compound is composed predominantly of amino acids/amino acid sequences, cyclization may be N- to C-terminal, N-terminal to side chain and N-terminal to backbone, C-terminal to side chain, C-terminal to backbone, side chain to backbone and side chain to side chain, as well as backbone to backbone cyclization. Cyclization of the compound may also take place through the non-amino acid organic moieties.
The association between the amino acid sequence component of the compound and other components of the compound may be by covalent linking, by non-covalent complexion, for example, by complexion to a hydrophobic polymer, which can be degraded or cleaved producing a compound capable of sustained release; by entrapping the amino acid part of the compound in liposomes or micelles to produce the final compound of the invention. The association may be by the entrapment of the amino acid sequence within the other component (liposome, micelle) or the impregnation of the amino acid sequence within a polymer to give the final compound of the invention.
Preferably the compounds comprise an amino acid sequence of (a) to (i) above in association with (in the meaning described above) a moiety for transport across cellular membranes.
The term xe2x80x9cmoiety for transport across cellular membranesxe2x80x9d refers to a chemical entity, or a composition of matter (comprising several entities) that causes the transport of members associated (see above) with it through phospholipidic membranes. One example of such moieties are hydrophobic moieties such as linear, branched, cyclic, polycyclic or heterocyclic substituted or non-substituted hydrocarbons. Another example of such a moiety are short peptides that cause transport of compounds attached to them into the cell by gradient derived, active, or facilitated transport. Other examples of other non-peptidic moieties known to be transported through membranes, such as glycosylated steroid derivatives, are well known in the art. Yet another example are moieties that are endocytosed by cellular receptors such as ligands of the EGF and tranferrin receptors. The moiety for the transport across membranes may be a polymer, liposome or micelle containing, entrapping or incorporating the amino acid sequence therein. In the above examples, the compound of the invention is the polymer, liposome, micelle, etc., impregnated with the amino acid sequence.
The term xe2x80x9ca sequence which is a continuous stretch of at least 5 amino acids present . . . xe2x80x9d refers to any continuous stretch of at least 5 amino acids, which are present in a longer amino acid sequence described by reference to positions of TGF-xcex2I (see below). For example, if in a specific TGF-xcex2 Ser/Thr kinase receptor, the positions corresponding to amino acid residues X to Y of TGF-xcex2I are amino acid residues 200 to 219 of that specific kinase, the continuous stretch of at least 5 amino acids may be from amino acid at position 200 to 204, from 201 to 205, from 216 to 220, from 210 to 214, etc. The continuous sequence may also be of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 amino acids, obtained from each of these positions.
The term xe2x80x9cTGF-xcex2 superfamily Ser/Thr kinase receptorxe2x80x9d (hereinafter xe2x80x9cTGF-xcex2 kinasexe2x80x9d) refers to both Type I receptors (ALK 1-7) as well as Type II receptors (ACTR-II, ACTR-IIb, TGF-B-II, BMPR-II, and AMHRII), and includes receptors which are activated by all of the ligands of the superfamily, examples of the ligands being TGF-xcex2, BMP""s, as activins/inhibins, AMH, and GDF. In one specific example, the TGF-xcex2 superfamily Ser/Thr kinase receptors are selected from ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, TGF-B-II, activin RII, BMPRII and ILK.
The term xe2x80x9csequence corresponding to positions . . . to . . . of TGF-xcex2Ixe2x80x9d refers to a sequence that matches the sequence appearing in the native TGF-xcex2I receptor 1 when the sequence of the catalytic unit of the specific kinase is aligned with the sequence of the catalytic unit of TGF-xcex2I. TGF-xcex2I is identified as in the chain-D crystal structure of the cytoplasmic domain of TGF-xcex2 receptor in complex with Fkbp12 (gi 5542077 in the NCBI database, in the PDB entry). The positions of the TGF-xcex2I receptor 1 kinase beginning in a certain position and ending in another position are indicated in the definition (a) to (d) above. For determining the beginning and end positions of the specific kinase used, the sequence of the catalytic unit of the specific kinase should be aligned with the sequence of the catalytic unit of TGF-xcex2I in pair-wise or multiple alignment manner. Alignment may be carried out using any state of the art software such as ClustAl(trademark) (version W or X).
FIG. 12 shows the alignment results of various kinases of the TGF superfamily Ser/Thr kinase receptor, in the above four regions, with TGF-xcex2I. Of course where the kinase is TGFxcex21 itself the positions are given, and there is no need for alignment.
The term xe2x80x9cwherein up to 40% of amino acids of the native sequence have been replaced with a naturally or non-naturally occurring amino acid or with a peptidomimetic organic moietyxe2x80x9d in accordance with the present invention, concerns an amino acid sequence, which shares at least 60% of its amino acid with the native sequence as described in (a), (b), (c) or (d) above, but some of the amino acids were replaced either by other naturally occurring amino acids, (both conservative and non-conservative substitutions), by non-naturally occurring amino acids (both conservative and non-conservative substitutions), or with organic moieties which serve either as true peptidomimetics (i.e., having the same steric and electrochemical properties as the replaced amino acid), or merely serve as spacers in lieu of an amino acid, so as to keep the spatial relations between the amino acid spanning this replaced amino acid. Guidelines for the determination of the replacements and substitutions are given in the detailed description part of the specification. Preferably no more than 30%, 25% or 20% of the amino acids are replaced.
The term xe2x80x9cwherein up to 40% of the amino acids have their side chains chemically modifiedxe2x80x9d refers to a variant which has the same type of amino acid residue, but to its side chain a functional group has been added. For example, the side chain may be phosphorylated, glycosylated, fatty acylated, acylated, iodinated or carboxyacylated. Other examples of chemical substitutions are known in the art and given below.
The term xe2x80x9cup to 20% of the amino have been deletedxe2x80x9d refer to an amino acid sequence which maintains at least 80% of its amino acids. Preferably no more than 10% of the amino acids are deleted and more preferably none of the amino acids are deleted.
The term xe2x80x9cprovided that at least 50% of the amino acids in the parent protein are maintained unaltered in the variantsxe2x80x9d, the up to 40% substitution, up to 40% chemical modification and up to 20% deletions are combinatorial, i.e., the same variant may have substitutions, chemical modifications and deletions so long as at least 50% of the native amino acids are identical to those of the native sequence both as regards the amino acid and its position. In addition, the properties of the parent sequence, in modulating tissue remodeling (for example as determined by TGF-xcex2 associated signal transduction), have to be maintained in the variant, typically at the same or at a higher level.
When calculating 40% (or 35, 30, 25, 20%) replacements, or 20% (or 10%) deletions from sequences, the number of actual amino acids should be rounded mathematically, so that 40% of an 11-mer sequence (4.4) is four amino acids and 40% of a 12-mer sequence (4.8) is five amino acids.
Typically xe2x80x9cessential amino acidsxe2x80x9d are maintained or replaced by conservative substitutions while non-essential amino acids may be maintained, deleted or replaced by conservative or non-conservative replacements. Generally, essential amino acids are determined by various Structure-Activity-Relationship (SAR) techniques (for example amino acids when replaced by Ala cause loss of activity) are replaced by conservative substitution while non-essential amino acids c an b e deleted or replaced by any type of substitution. Guidelines for the determination of the deletions, replacements and substitutions are given in the Detailed Description part of the specification.
The term xe2x80x9cregionxe2x80x9d refers to a sequence in a specific location in the specific member of the TGF-xcex2 superfamily Ser/Thr kinase receptor which corresponds to the positions selected from: 249 to 279 of TGF-xcex2I (termed: HJ loop); positions 119 to 139 (termed: xcex1D loop); positions 104 to 115 (termed: B4-B5 region); and positions 89 to 103 (termed: A-loop).
The term xe2x80x9ccorresponding D-amino acidxe2x80x9d refers to the replacement of the naturally occurring L-configuration of the natural amino acid residue by the D-configuration of the same residue.
The term xe2x80x9cat least one peptidic backbone has been altered to a non-naturally occurring peptidic backbonexe2x80x9d means that the bond between the N- of one amino acid residue to the C- of the next has been altered to non-naturally occurring bonds, for example, by reduction (to xe2x80x94CH2xe2x80x94NHxe2x80x94), alkylation (methylation) on the nitrogen atom, or the bonds have been replaced by amidic bonds, urea bonds, or sulfonamide bonds, etheric bonds (xe2x80x94CH2xe2x80x94Oxe2x80x94), thioetheric bonds (xe2x80x94CH2xe2x80x94Sxe2x80x94), or to xe2x80x94CSxe2x80x94NHxe2x80x94. The side chain of the residue may be shifted to the backbone nitrogen to obtain N-alkylated-Gly (a peptidoid).
The term xe2x80x9cin reverse orderxe2x80x9d refers to the fact that the sequence of (a) to (f) may have the order of the amino acids as it appears in the native TGF-xcex2 kinase from N- to the C-direction, or may have the reversed order (as read in the C- to N-direction). For example, if a subsequence of the A-region of TGFxcex2 receptor is QTVML (residues 1-5 of SEQ ID NO:53) a sequence in a reverse order is LMVTQ (SEQ ID NO:60). It has been found that many times sequences having such a reverse order can have the same properties, in small peptides, as the xe2x80x9ccorrectxe2x80x9d order, probably due to the fact that the side chains, and not the peptidic backbones, are those responsible for interaction with other cellular components. Particularly preferred are what is termed xe2x80x9cretro inversoxe2x80x9d peptidesxe2x80x94i.e., peptides that have both a reverse order as explained above, and in addition each and every single one of the amino acids has been replaced by the non-naturally occurring D-amino acid counterpart, so that the net end result, as regards the positioning of the side chains (the combination of reverse order and the change from L to D), is zero change. Such retro-inverso peptides, while having similar binding properties to the native peptide, were found to be resistant to degradation.
The present invention further concerns a method for the modulation of tissue-remodeling in a subject, comprising administering to a subject in need of such treatment a therapeutically effective amount of a compound comprising a sequence selected from:
(a) a sequence which is a continuous stretch of at least five amino acids present in a native TGF-xcex2 superfamily Ser/Thr kinase receptor, in positions of the kinase corresponding to positions 249 to 279 of TGF-xcex2 Receptor 1) (HJ loop);
(b) a sequence which is a continuous stretch of at least five amino acids present in a native TGF-xcex2 superfamily Ser/Thr kinase receptor, in positions of the kinase corresponding to positions 119 to 139of TGF-xcex2 Receptor 1 (xcex1D region);
(c) a sequence which is a continuous stretch of at least five amino acids present in a native TGF-xcex2 superfamily Ser/Thr kinase receptor, in positions of the kinase corresponding to positions 104 to 115 of TGF-xcex2 Receptor 1 (B4-B5 region);
(d) a sequence which is a continuous stretch of at least five amino acids present in a native TGF-xcex2 superfamily Ser/Thr kinase receptor, in positions of the kinase corresponding to positions 89 to 103 of TGFxcex2 TGF-xcex2 Receptor 1 (A-region);
(e) a variant of a sequence according to any one of (a) to (d) wherein up to 40% of the amino acid of the native sequence have been replaced with a naturally or non-naturally occurring amino acid or with a peptidomimetic organic moiety; and/or up to 40% of the amino acids have their side chains chemically modified; and/or up to 20% of the amino acids have been deleted; provided that at least 50% of the amino acids in the parent sequence of (a) to (d) are maintained unaltered in the variant, and provided that the variant maintains the biological activity of the parent sequences of (a) to (d);
(f) a sequence of any one of (a) to (e) wherein at least one of the amino acids is replaced by the corresponding D-amino acid;
(g) a sequence of any one of (a) to (f) wherein at least one of the peptidic backbones has been altered to a non-naturally occurring peptidic backbone;
(h) a sequence being the sequence of any one of (a) to (g) in reverse order; and
(i) a combination of two or more of the sequences of (a) to (h).
The term xe2x80x9csubjectxe2x80x9d in the meaning of the present invention concerns a human, but can also include animals for veterinary purposes.
The term xe2x80x9ctherapeutically effective amountxe2x80x9d is the quantity of compound which results in an improved clinical outcome as a result of the treatment compared with a typical clinical outcome in the absence of the treatment. An xe2x80x9cimproved clinical outcomexe2x80x9d results in an individual with the disease experiencing fewer symptoms or complications of the disease, including a longer life expectancy, as a result of the treatment as well as the prevention of the disease before it occurs. With respect to bones, an xe2x80x9cimproved clinical outcomexe2x80x9d includes, but is not limited, to a shorter time period until bone healing is achieved; or to the healing of bone in cases where normal bone healing does not take place such as where a large bone section is missing (xe2x80x9cnon-unionxe2x80x9d). With respect to post-surgical fibrosis or scarring, the term refers to smaller amount fibrosis or scarring or prevention of the formation of either one before they are expected to occur, such as prevention of scarring or fibrosis after surgery. With respect to alopecia it concerns elimination or decrease in the amount of hair-loss in general and in particular following chemotherapeutic or radiotherapeutic treatment. The amount of compounds of the invention administered to the individual will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs as well as on the mode of administration. In the case of tissue remodeling, many applications are local to the tissue, the amounts used when locally administered may be smaller than in systemic administration. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. Typically, a therapeutically effective amount of the compound can range from about 1 mg per day to about 1000 mg per day for an adult. Preferably, the dosage ranges from about 1 mg per day to about 100 mg per day.
The method for therapeutic treatment, by way of tissue remodeling may be used for improving bone healing (including improvement in rate of healing, achievement of healing in cases where a large bone segment xe2x80x9cnon-unionxe2x80x9d is missing, augmentation of various bones, increasing the probability of obtaining healthy bones); decreasing fibrosis (as determined by decreased scarring, decreased adhesion, decreased deposition of collagen and extracellular matrix in various diseases and post-operational and injury including injury to the skin and connective tissue), decreased alopecia and the like.
The present invention also concerns methods for obtaining the above compounds which can be used in a method for modulation of tissue remodeling. Thus the present invention concerns a method for obtaining a compound for the modulation of tissue-remodeling the method comprising:
(i) providing a plurality of candidate compounds comprising the sequences as defined above;
(ii) assaying the candidate compounds obtained in (i) in a test assay and determining the level of tissue-remodeling;
(iii) selecting those compounds which modulate tissue-remodeling as compared to the tissue-remodeling in the same test assay in the absence of the candidate compounds, thereby obtaining compounds being capable of modulating tissue modulating activities.
Assays for determining tissue-modeling activity will be specified in more detail hereinafter below. However, many of these assays require testing in vivo, or in tissue cultures, and thus are rather expensive and complicated for initial screening purposes. Therefore, many times it is preferable to conduct initial screening in a cellular, or cell-free system, which is used to determine the level of TGF-xcex2 superfamily Ser/Thr kinase receptor signal transduction pathways. Examples of such systems are assays for determination of the number of cells, which proliferation is dependent on this pathway, measurement of collagen deposition by cells, determination of level of phosphorylation of the substrate of the TGF-xcex2 kinase in intact cells or in cell free systems and the like. Compounds which are capable of modulating a TGF-xcex2 associated signal transduction have a high probably of also being active in tissue remodeling, however, this activity still has to be verified in a more relevant physiological assays.
Typically, modulation of the level of TGF-xcex2 associated signal transduction refers to a change in the level of phosphorylation of at least one the TGF-xcex2 substrate (which may be the direct or indirect substrate.
It should be appreciated that for the purpose of modulation, it is best to choose a compound comprising sequences derived from the same member of the TGF-xcex2 superfamily Ser/Thr kinase receptor as the one known (for example, in literature or from clinical information) to be involved in the modulation of the specific tissue of interest.
It should be appreciated that some of compounds comprising the sequences of (a) to (i) above, have better tissue remodeling modulating activities than others, and the selection of the compounds which are active in the tissue remodeling process should be done according to the method as indicated above.
Preferably, the determination of the sequence to be included in the candidate compound for modulating tissue remodeling should be carried out with the following steps:
(i) determining which specific member of the TGF-xcex2 superfamily Ser/Thr kinase receptor is involved in the remodeling of the tissue to be modulated, and determining the sequence of the specific member from a database of amino acid sequences;
(ii) aligning the sequence of the catalytic unit of the member obtained in (i) with the sequence of the catalytic unit of TGF-xcex2I receptor, and determining the sequence of the specific member in four regions corresponding, in the alignment, to the following, positions of TGF-xcex2I: 249 to 279 (HJ-loop), 119 to 139(xcex1D region), 104 to 115 (B4-B5 region), 89 to 103 (A-region);
(iii) determining a continuous stretch of at least 5 amino acids of any of the four regions of (ii) above, that is sorter than the length of the entire region and has modeling activities of the tissue-remodeling or TGF-xcex2kinase associated signal transduction, by: synthesizing a plurality of subsequences (optionally partially overlapping subsequences) of 5-10 mer from any of the above four regions; testing those sequences in a test assay for determining tissue-remodeling or TGF-xcex2-associated signal transduction modulating activities, and selecting those sequences that have tissue remodeling or TGF-xcex2-associated signal transduction modulating activities;
(iv) determining in the sequences of (ii) or in the sequences selected in (iii) above, essential and non-essential amino acids by: preparing a plurality of modified sequences wherein in each modified sequence a single and different position in the native sequence has been replaced with a test amino acid (preferably with Ala); testing those modified sequences in a test assay to determine tissue-remodeling or TGF-xcex2-associated signal transduction modulating activities; those amino acids which when replaced caused a statistically significant change in tissue-remodeling TGF-xcex2-associated signal transduction modulating activity being essential amino acids, and those amino acids which when replaced, did not cause a statistically significant change in tissue remodeling/TGF-xcex2-associated signal transduction modulating activity, being non-essential amino acids;
(v) preparing a plurality of compounds comprising sequences selected from:
(1) the sequences of (ii);
(2) the sequences selected in (iii);
(3) the sequences of (ii) or the selected sequence of (iii), wherein at least one of the essential amino acids has been replaced by a conservatively substituted naturally or non-naturally occurring amino acid, or a conservative peptidomimetic organic moiety; and/or at least one of the non-essential amino acids has been deleted, or substituted (conservatively or non-conservatively) by naturally or non-naturally occurring amino acids or a peptidomimetic;
(4) the sequences of (1) to (3) in a reverse order;
(5) the sequence of (4) wherein all the amino acids have been replaced by their D-counterpart residues;
said compounds of (v) being candidate compounds for modulating tissue remodeling.
Conceptually, the first step is deciding which specific member of the TGF-xcex2 superfamily Ser/Thr kinase receptor is involved in the tissue-remodeling which is to be modulated. This can be done for example by carrying out a literature search, and determining which kinase is known to be involved in the processes of tissue remodeling that is to be modulated.
Once this specific kinase is identified, its sequence can be obtained from amino acid sequence databases and it is possible to locate the above four regions, simply by aligning the sequence of the catalytic unit of the specific kinase chosen, as present in the database, with the TGF-xcex2 Receptor I catalytic subunit, and by this finding the specific sequences of the four regions. Although the sequences of the region are not very long (12-31 amino acids long), it is of course desirable to find the shorter subsequence of at least 5 continuous amino acids present within this full region, and use this shorter sequence in the compound of the invention. Finding this short subsequence is a routine procedure, which can be achieved by several possible manners, such as by synthesizing sequences of 5-10 mer having partially overlapping, or adjacent sequences, and optionally optimizing the chosen sequence (if rather longer sequences such as, for example, 8-10 mer are used) by sequentially deleting from one or both of its terminal amino acids until the optimal shorter sequence (not necessarily the shortest but a combination of length and activity should be considered) sequence still having tissue-remodeling modulating activities is obtained.
After obtaining shorter subsequence which still has tissue-remodeling properties (as may be determined both by a tissue-remodeling assay or in an indicative assay such as modulation of TGF-xcex2-kinase associated signal transduction), it is necessary to find which amino acids either in the sequence of the full region but preferably in the sequence of the shorter subsequence are essential (crucial for the modulating activity) and which are non-essential. This can be done by routine procedure, wherein a plurality of sequences are prepared, wherein in each sequence a single (and different) amino acid has been replaced, as compared to the native sequence by a xe2x80x9ctest amino acidsxe2x80x9dxe2x80x94 usually the amino acid residue Alanine (a procedure known as: xe2x80x9cAla-scanxe2x80x9d). Each of the plurality of sequences is again tested for its tissue-remodeling/TGF-xcex2-associated signal transduction modulating activities. Amino acids which when replaced cause lost, or substantial decrease in the modulating activity of the full sequence is considered as xe2x80x9cessential amino acidsxe2x80x9d. Amino acids which when replaced do not caused a change of modulating activity of the sequence are referred to as xe2x80x9cnon-essentialxe2x80x9d amino acids (the loss or decrease should be determined by statistically significant manners).
Finally, as a last step, a plurality of sequences is prepared which may comprise either the full native sequence of any of the regions, short subsequence of at least 5 amino acids as appearing in any of the regions, sequences wherein at least one essential amino acid has been replaced by conservative substitution by a naturally, non-naturally occurring amino acid or by a peptidomimetic organic moiety; and/or an amino acid sequence wherein at least one amino acid (present in a non-essential position) has been deleted, or an amino acid in a non-essential position has been replaced by conservative or non-conservative substitution by a naturally occurring, non-naturally occurring, or organic peptidomimetic moiety.
For example, 1, 2, 3, 4, 5, 6, 7, 8, amino acids may be replaced in the sequence used in the compound of the invention as compared with the native sequence present in the kinase. The total combination of replacements, deletions, etc. should be such that the resulting variant host where at least 50% of the amino acids of the native sequence are present unaltered.
A notable exception to the above is the use of retro-inverso amino acids (in reverse order as compared to the native sequence), where when the peptide is in the reversed order, all of its amino acids are replaced with their D-counterparts.
When preparing the compound, it is possible to proceed by one of two strategies: by one strategy it is possible to test (for tissue remodeling or TGF-xcex2-associated signal transduction modulating activities) a full compoundxe2x80x94i.e., a compound comprising both a candidate sequence, and for example, non-amino acid moieties such as hydrophobic moieties present in one of its terminals. This strategy is generally used where the test assay is intact cells or in-vivo where the issue of penetration through membranes, addressed by addition of a hydrophobic moiety, is crucial.
Alternatively, it is possible to first optimize the sequence alone (preferably by testing it in a cell-free system for TGF-xcex2-signaling dependent phosphorylation) so as to first find the best sequence possible, and then add to the optimal sequence other moieties, such as hydrophobic moieties, etc. to improve other properties of the compound as a whole such as for improving the penetration into cells, resistance to degradation, etc.
The present invention also concerns compounds for modulation of tissue-remodeling obtained by any of the above methods.
The present invention further concerns pharmaceutical compositions comprising the above compounds as active ingredients. The pharmaceutical composition may contain one species of compounds of the invention or a combination of several species of the invention.
The pharmaceutical compositions of the invention should be used for treatment of conditions or disorders wherein a therapeutically beneficial effect can be evident through the modulation of tissue remodeling.